BZM055, an Iodinated Radiotracer Candidate for PET and SPECT Imaging of Myelin and FTY720 Brain Distribution

Article abstract of DOI:10.1002/cmdc.201000477

FTY720 (fingolimod, Gilenya) is a sphingosine 1-phosphate (S1P) receptor modulator that shows significant therapeutic efficacy after oral administration to patients of multiple sclerosis. Because FTY720 does not contain any atom whose PET or SPECT radioisotope would have a half-life compatible with its pharmacokinetic properties, it cannot be used directly for imaging. Instead, we propose BZM055 as a surrogate tracer to study its pharmacokinetics and organ distribution in patients and, given that FTY720 accumulates in myelin sheaths, for myelin imaging. BZM055 (2a, 2-iodo-FTY720) can be easily radiolabeled with ¹²³I (for SPECT) or ¹²⁴I (for PET). Not only does it closely mimic the pharmacokinetics and organ distribution of FTY720, but also its affinity, selectivity for S1P receptors, phosphorylation kinetics, and overall physicochemical properties. [¹²³I]BZM055 is currently under development for clinical imaging.

Full text of DOI:10.1002/cmdc.201000477

DOI: 10.1002/cmdc.201000477
BZM055, an Iodinated Radiotracer Candidate for PET and
SPECT Imaging of Myelin and FTY720 Brain Distribution
Emmanuelle Briard,*[a] David Orain,[a] Christian Beerli,[b] Andreas Billich,[b] Markus Streiff,[b]
Marc Bigaud,[b] and Yves P. Auberson[a]
FTY720 (fingolimod, Gilenyaꢀ) is a sphingosine 1-phosphate
(S1P) receptor modulator that shows significant therapeutic ef-
ficacy after oral administration to patients of multiple sclerosis.
Because FTY720 does not contain any atom whose PET or
SPECT radioisotope would have a half-life compatible with its
pharmacokinetic properties, it cannot be used directly for
imaging. Instead, we propose BZM055 as a surrogate tracer to
study its pharmacokinetics and organ distribution in patients
and, given that FTY720 accumulates in myelin sheaths, for
myelin imaging. BZM055 (2a, 2-iodo-FTY720) can be easily ra-
diolabeled with 123I (for SPECT) or 124I (for PET). Not only does it
closely mimic the pharmacokinetics and organ distribution of
FTY720, but also its affinity, selectivity for S1P receptors, phos-
phorylation kinetics, and overall physicochemical properties.
[
123I]BZM055 is currently under development for clinical imag-
ing.
Introduction
FTY720 (fingolimod, Gilenyaꢀ, Scheme 1 below) is a sphingo-
sine 1-phosphate (S1P) receptor modulator that has shown sig-
nificant improvement in relapse rate and MRI outcomes after
oral administration to patients of multiple sclerosis (MS).[1]
FTY720 prevents lymphocyte egress from lymph nodes,[2] lead-
ing to decreased infiltration of lymphocytes into the central
nervous system (CNS).[3] Preclinical evidence also suggests that
it may provide additional neuroprotection to the CNS by mod-
ulation of cerebral S1P receptors.[4,5]
lation rates of tracer candidates, we also synthesized their
phosphate analogues to enable comparison of their affinity for
S1P receptors, and to adequately compare their overall proper-
ties with AML629. After all candidate radiotracers were profiled
according to these criteria, we selected BZM055 (2a) as the
closest mimic of the drug for imaging studies.
To further study the therapeutic action of FTY720, it would
be useful to quantify its pharmacokinetics and organ distribu-
tion in patients. As it has already been shown in rodents that
FTY720 accumulates in myelin sheaths,[6] we reasoned that an
imaging agent based on this drug might also allow myelin
imaging and the study of disease progression, in addition to
following the pharmacokinetics of FTY720 in the brain tissues
of MS patients.
Results and Discussion
Chemistry
The most common radioisotopes for PET imaging are 18F and
11C. However, their short half-lives (109.8 and 20.3 min, respec-
tively) are not compatible with the distribution kinetics of
FTY720 in humans. The alternative approach, using the side
chain of FTY720 to attach a chelating group for 64Cu was not
considered, as this would have modified its properties too
much. Instead, we elected to prepare iodinated derivatives of
FTY720. Iodine-123, with a radioactivity half-life of 13.2 h, is
widely used for SPECT. In contrast, iodine-124 has a radioactive
half-life of 4.2 days and is used for PET. This makes iodine de-
rivatives well-suited candidates for imaging FTY720, the
plasma half-life of which in human subjects is six days.[8] Alter-
FTY720 does not contain any atom whose positron emission
tomography (PET) or single photon emission computed to-
mography (SPECT) radioisotope would have a half-life compati-
ble with its pharmacokinetic properties. It is therefore not pos-
sible to study this drug with such imaging techniques without
modifying it slightly. With this in mind, we defined the criteria
that would make a radiolabeled derivative of FTY720 an ade-
quate surrogate of the drug, mimicking its pharmacokinetics
and organ distribution, while retaining affinity and selectivity
for S1P receptors, as well as having similar overall physico-
chemical properties.
[a] Dr. E. Briard, Dr. D. Orain, Dr. Y. P. Auberson
Novartis Institutes for BioMedical Research, Basel
Global Discovery Chemistry, Neurosciences and Molecular Imaging
Postfach, 4002 Basel (Switzerland)
FTY720 itself is inactive at S1P receptors, but is phosphory-
lated in vivo by sphingosine kinase-2 (SphK2) to (S)-FTY720
phosphate (AML629), which is the active moiety that binds to
S1P receptors.[2b,7] As a consequence, any derivative that
mimics FTY720 should be a substrate of SphK2 and phos-
phorylated in a similar manner. Besides measuring phosphory-
Fax: (+41)616-962-455
E-mail: emmanuelle.briard@novartis.com
[b] Dr. C. Beerli, Dr. A. Billich, Dr. M. Streiff, Dr. M. Bigaud
Novartis Institutes for BioMedical Research, Basel
Autoimmunity, Transplantation and Inflammation Disease Area
Postfach, 4002 Basel (Switzerland)
ChemMedChem 2011, 6, 667 – 677
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667

E. Briard et al.
MED
natively, 76Br, which decays with a half-life of 16.1 h, would be
iodination and enhance chemical stability, these derivatives
were prepared as vinyl iodides.[13]
a possible alternative for PET imaging.
Based on available structure–activity information, we fo-
cused on two options to introduce an iodine atom on the
FTY720 scaffold.[9] The first and more straightforward approach
is by aromatic iodination (Scheme 1), which we expected to
maintain affinity to S1P receptors based on closely related de-
rivatives.[10] This iodination was also performed on 1,[9] an equi-
potent alkyl ether analogue of FTY720.
It was not possible to directly convert the terminal acetylen-
ic group of an FTY720 analogue to an iodovinylic derivative, as
an incomplete and unselective iodination led to inseparable
mixtures. Instead, we started from isomerically pure iodovinylic
building blocks as depicted in Scheme 3. Compounds 6a, 6b,
and 6c were prepared by following modified published proce-
dures,[14,15] followed by treatment under high vacuum to
remove contaminating hex-6-yn-1-ol and hex-6-en-1-ol. Mitsu-
nobu condensation with 7[16] yielded compounds 8a–c, which
were then deprotected under acidic conditions to avoid iodo-
lysis, yielding FTY720 iodovinyl analogues 9a–c.
Phosphorylation of 8a–c was carried out under standard
conditions, with racemic material rather than after optical reso-
lution, as racemization of the pure enantiomers of 8a–c had
been observed at room temperature. Compounds 10a–c were
isolated as mixtures with hydroxylated methyl oxazoles as by-
products. They were used in the next step without further pu-
rification. Deprotection under acidic conditions yielded iodovi-
nylic phosphonates 11 a–c as off-white powders. Because it
was not possible to separate the enantiomers of 11 a–c using
classical Chiralpack AS, those were further characterized as rac-
emic mixtures.
Scheme 1. Iodinating FTY720 and its ether analogue 2: a) I2, Ag2SO4,
CF3SO3Ag, RT, 18 h in CH2Cl2/CH3OH: 2a, 2b 60% or wet CH2Cl2: 2c 64%.
FTY720 was iodinated by using iodine and silver sulfate in
the presence of a catalytic amount of silver trifluoromethane-
sulfonate[11] to give a 2:1 mixture of 2a and 2b in 60% yield.
The two regioisomers were separated by supercritical fluid
chromatography. In contrast, compound 2c was obtained as a
single regioisomer in 64% yield, starting from aryl alkyl ether
1.
The synthesis of the corresponding chiral phosphates 5a–5c
is outlined in Scheme 2: After protection of 2a–2c as oxazo-
lidinones 3a–c, phosphorylation with di-tert-butyl-N,N-diethyl-
phosphoramidite, oxidation with tBuOOH, and subsequent
enantiomeric separation using Chiralpack AS yielded the pro-
tected phosphate esters 4a–c. Hydrolysis with lithium hydrox-
ide and hydrochloric acid gave the desired phosphates 5a–c.
The second approach to iodinated FTY720 derivatives intro-
duces a halogen atom on the aliphatic side chain (Scheme 3).
It is known that the latter can be modified relatively broadly
while maintaining affinity for S1P receptors.[12] To prevent de-
Profiling
The addition of an iodine atom to FTY720 has a significant
effect on its molecular weight, corresponding to a mass in-
crease of 41% (33% for the phosphate). It also significantly in-
creases lipophilicity, as indicated by the logD (pH 7.4) values of
2a and 2b, relative to FTY720 (Table 1). Other compounds
listed in this table have lower logD values, resulting from an
oxygen atom inserted in the side chain. Interestingly, despite
these changes the affinity for S1P receptors (Table 2) was
hardly altered, except for a decrease in S1P3 affinity.
Relative to the natural substrate d-sphingosine, FTY720 is
phosphorylated by human recombinant SphK2 at a rate of
14.4% relative to d-sphingosine.[7] The rates for the iodinated
derivatives are similarly low, ranging from 2.4 to 9% (Table 1).
Overall, this conversion remains rapid relative to the distribu-
tion kinetics of both species
in vivo,[8] and minor differences
are not expected to have an in-
fluence on imaging properties.
FTY720 is mainly metabolized
in the liver by hydroxylation and
oxidation of the alkyl chain,[17]
and it was previously shown
that a rat PBPK model can pre-
dict human PK.[6b] We therefore
used in vitro clearance measure-
[18]
ments (CL’int
) in rat micro-
somes (Table 1) to compare the
anticipated metabolic rates of
2a–c and 9a–c. These results
show that iodophenyl deriva-
Scheme 2. Phosphorylation of iodinated FTY720 derivatives: a) benzylchloroformate, NaOH, RT, 12 h: 3a 75%, 3b
77%, 3c 78%; b) (tBuO)2PN(Et)2, 1H-tetrazole, CH2Cl2/THF, RT, 18 h; c) tBuOOH, RT, 90 min; d) Chiralpak separation;
e) LiOH·H2O, 608C, 20 h; f) HCl, RT, 1 h: (S)-5a 32%, (S)-5b 63%, (S)-5c 57%.
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ChemMedChem 2011, 6, 667 – 677

BZM055
Scheme 3. Synthesis of iodovinylic FTY720 analogues 11 a–c: a) 6a: 1) Cp2ZrCl2/LiEt3BH, THF, 2) N-iodosuccinimide, 3) AcOH/TEA (overall yield 19%); 6b:
1) ref. [14], 2) AcOH/TEA; 6c: ref. [15]; b) DIAD, PPh3, THF, RT: 8a 59%, 8b 53%, 8c 55%; c) 33% HCl(aq), EtOH, 858C, 2.5 h: 9a 78%, 9b 33% or 33% HCl(aq)
,
dioxane, 508C, 20 h, then 708C, 4 h: 9c 54%; d) 1) 1H-tetrazole, (tBuO)2P(O)O, CH2Cl2, 6 h, RT, 2) 30%wt H2O2, 90 min, RT: 10a 32% (50% purity), 10b 98%
(70% purity), 10c 80% (70% purity); e) 33% HCl(aq), EtOH, 858C, 2.5 h: 11 a 84%, 11 b 42% or 33% HCl(aq), dioxane, 508C, 4 h: 11 c 30%.
FTY720. They are also the compounds with the greatest struc-
Table 1. Profile of iodinated derivatives of FTY720 and their phosphory-
lated analogues.
tural differences from the drug, and possibly the most chal-
lenging radiochemistry. As a consequence, they do not seem
to offer any advantage over other derivatives. We hence select-
ed 2a and 2b, which have the closest profile to FTY720, for
further evaluation.
Parent
logD[a]
Mr
[Da]
PSA
[ꢂ2]
Phos.
Rate [%][b]
CL’int
[c]
[mLminꢀ1 kgꢀ1
]
FTY720
2a (BZM055)
1.9
2.9
2.8
2.0
0.9
1.0
0.7
307
433
433
435
419
419
419
66.5
66.5
66.5
75.7
75.7
75.7
76
14.4ꢁ1.2
9.0ꢁ0.5
5.6ꢁ0.3
2.7ꢁ0.15
2.4ꢁ0.2
2.7ꢁ0.2
6.0ꢁ0.4
13.7
30.4
5.5
<5.5
34.3
48.8
38.5
As a next step, and because PET tracers are usually given in-
travenously, we compared the rat PK/PD profiles of these two
tracer candidates with those of FTY720, after i.v. administration.
Compound 2a (BZM055) shows a concentration profile similar
to that of FTY720 in blood. In the brain, its profile also follows
the same kinetics, although with a 2.8-fold lower exposure.
Phosphate 5a has similar kinetics to AML629 in blood and
brain, equally with a lower exposure, possibly due to a slightly
larger distribution volume (Figure 1).
2b
2c
9a
9b
9c
[a] Measured by HPLC. [b] Phosphorylation rate relative to S1P; n=3.
[c] In vitro clearance measured with rat liver microsomes.
Compound 2b has similar properties to FTY720 as well;
however, the blood AUC of its phosphate 5b is proportionally
lower. Furthermore, 5b only penetrates the brain to a limited
extent: With a brain-to-blood AUC ratio of 0.2, it differs signifi-
cantly from AML629 and 5a, which have ratios of 1.4 and 1.2,
respectively (Table 3). This indicates that although 2b might be
a valuable tool to quantify the contribution of brain penetra-
tives 2b and especially 2c have lower clearance than FTY720,
whereas clearance is higher for 2a and iodovinyl derivatives
9a–c.
Compound 2c has properties that clearly differ from FTY720,
especially with regards to its very low clearance. In contrast,
the iodovinyl derivatives 9a–c are metabolized faster than
Table 2. Affinity and maximal efficacy values for S1P receptors .
S1P1
EC50 [nm][a]
S1P3
EC50 [nm][a]
S1P4
EC50 [nm][a]
S1P5
EC50 [nm][a]
Phosphates
Mr [Da]
Emax [%][b]
Emax [%][b]
Emax [%][b]
Emax [%][b]
AML629
5a
5b
387
513
513
515
499
499
499
0.17ꢁ0.067 (10)
0.6ꢁ0.37 (3)
0.2ꢁ0.12 (3)
0.8ꢁ0.2 (3)
1.7ꢁ1.4 (2)
3.2ꢁ1.6 (3)
3.5ꢁ2.6 (2)
96
104
107
96
93
89
2.5ꢁ0.9 (6)
47ꢁ25 (2)
85ꢁ78 (2)
33ꢁ16 (2)
7.5ꢁ2.5 (4)
11ꢁ11 (3)
11ꢁ2.2 (2)
97
44
28
31
60
67
66
0.9ꢁ0.33 (4)
7.1ꢁ3 (2)
68
98
0.58ꢁ0.32 (15)
1.6ꢁ0.35 (2)
2.1ꢁ0.5 (2)
1.1ꢁ0.5 (4)
0.6ꢁ0 (3)
64
72
87
66
62
68
59
5ꢁ2.1 (2)
118
136
127
114
118
5c
2.5ꢁ1.1 (3)
1.7ꢁ1 (4)
11 a
11 b
11 c
3.8ꢁ2.6 (2)
2.9ꢁ0.5 (3)
13ꢁ14 (3)
88
3.5ꢁ1.8 (3)
[a] Affinity values were estimated with a [g-35S]GTP binding assay; values represent the mean ꢁSEM (n). [b] Emax: maximal efficacy of analogue of S1P at
S1P1, S1P3, and S1P5 and AFD(R)[19] at S1P4.
ChemMedChem 2011, 6, 667 – 677
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669

E. Briard et al.
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Pharmacodynamic effects on lymphocyte depletion were
measured in the same experiment as the PK properties of
parent and phosphates, providing additional information on
the similarity of these molecules. As an example, when admin-
istered to rats at 4 mgkgꢀ1 i.v., BZM055 and 2b induced a simi-
lar decrease in circulating lymphocytes as did FTY720, with
similar kinetics and amplitudes. Their blood exposures induced
full efficacy on peripheral lymphocyte reduction over 48 h. In
contrast, the effect of 9c, an iodovinyl derivative tested for
comparison, started to subside toward the end of the observa-
tion period (Figure 2).
Figure 2. Kinetics of decreasing lymphocyte count in Lewis rats treated with
BZM055, 2b, and 9c versus FTY720 (each at 4 mgkgꢀ1 i.v., n=3).
Based on a similar PK profile to that of FTY720, relatively
good brain penetration, and a similar rate of phosphorylation,
BZM055 was selected for further development. The feasibility
of radiosynthesis of BZM055, under conditions that are com-
patible with PET radiochemistry, was confirmed by using stable
iodine (Scheme 4). The aim was to prepare the precursor that
would be used for radiolabeling in the SPECT or PET center
(compound 14), and to demonstrate that it could be converted
into BZM055 using reagents and conditions approaching those
that would be used with radioactive iodine.
Figure 1. Blood–brain distribution of a) amino alcohols and b) phosphates:
comparison of the time–concentration curves for BZM055 and 5a relative to
FTY720 and its phosphate AML629 after intravenous administration. For
technical reasons n=1 for the 0.5 and 4 h time points, for all others n=3;
error bars represent the mean ꢁSEM.
The precursor for radiolabeling was prepared from BZM055
itself, which was first protected with di-tert-butyldicarbonate
and 2,2-dimethoxypropane, yielding 12. This protected deriva-
tive was converted into pinaco-
tion to the therapeutic efficacy of S1P agonists, it would not
be an adequate surrogate of FTY720 for in vivo imaging.
lato boronate 13. However,
Table 3. PK parameters of FTY720, BZM055, 2b and their phosphates.
direct iodination of 13 as well as
of the corresponding neopentyl
Compd
FTY720
AUC0.5–48h [mm·h]
Cmax [mm]
tmax [h]
t1/2 [h]
Brain/Blood Ratio
boronate, was unsuccessful. We
therefore converted 13 into the
more reactive potassium trifluor-
oborate 14 using aqueous po-
tassium hydrogen fluoride.[20] In
a cold run approaching PET radi-
olabeling conditions, 14 was
converted back into BZM055 by
reaction with sodium iodide in
the presence of chloramine T in
~60% yield (10 mg scale). This
Blood
Brain
Blood
Brain
Blood
Brain
Blood
Brain
Blood
Brain
Blood
Brain
23.8
154.4
26.9
54.3
29.8
47.5
140.5
191.6
44.1
53.2
13.4
2.1
1.7
3.7
5.3
1.5
1.2
1.3
5.1
6.2
1.6
1.8
0.5
0.05
0.5
24
0.5
24
2
48
4
48
4
26.3
n.a.
12.2
n.a.
22.5
n.a.
23.6
n.a.
23.1
n.a.
27.1
n.a.
6.5
2.0
1.6
1.4
1.2
0.2
BZM055
2b
AML629
5a
48
8
48
5b
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BZM055
pled with a Bruker Hyperion 2000 microscope over a
wavenumber range of 4000–600 cmꢀ1, with a resolution
of 4 cmꢀ1. High-resolution and high-accuracy mass spec-
tra were acquired on a LTQ Orbitrap XL mass spectrome-
ter (Thermo Scientific) equipped with an electrospray ion
source operated in positive ion mode.
Analytical LC–MS/HPLC conditions (%=percent by
volume)
Method
A (tRA =retention time A): UPLC-ZQ2000,
Scheme 4. Synthesis of precursor 14 and iodination to BZM055 under conditions com-
patible with SPECT radiochemistry: a) Boc2O, NaOH, dioxane, RT, 18 h, 83%; b) 2,2-dime-
thoxypropane, acetone, pTsOH, DMF, RT, 20 h, 12 88%; c) bis(pinacolato)diboron, [PdCl2-
(dppf)], KOAc, DMSO, 808C, 6 h, 13 68%; d) aq.KHF2, CH3OH, RT, 15 min, 14 74%; e) NaI
(0.2 equiv), chloramine T, THF/H2O (1 mL), 508C, 2 h; f) HCl (6n), 508C, 1 h, 16 60%.
column: Acquity HSS-T3 1.8 mm, 2.1ꢃ50 mm; gradient:
A, H2O + 5% CH3CN + 0.5–1.0% HCO2H; B, CH3CN +
0.5–1.0% HCO2H; from 98:2 to 2:98 in 4.3 min
0.7 min isocratic; flow rate: 1.0 mLminꢀ1
+
.
Method B (tRB =retention time B): Gilson 331 pumps
coupled to a Gilson UV/Vis 152 detector and a Finnigan
AQA spectrometer (ESI), a 50 mL loop injection valve,
reaction was later used for the production of [123I]BZM055 for
and a Waters XTerra MS C18 3.5 mm 4.6ꢃ50 mm column running a
imaging studies.
gradient of H2O + 0.05% TFA/CH3CN + 0.05% TFA from 95:5 to
10:90 over 8 min with a flow rate of 1.5 mLminꢀ1
.
Conclusions
A series of six iodinated FTY720 analogues were synthesized
and profiled as potential surrogates of the drug, for imaging
purposes. Iodovinyl and 3-substituted derivatives proved to
differ from FTY720 in their properties, among others due to
lower brain penetration of their phosphate, or faster metabo-
lism. In contrast, 2-iodo-FTY720 (BZM055) is a promising tracer
candidate for studying the pharmacokinetics and organ distri-
bution of FTY720 in human subjects, as well as for imaging
myelin sheaths. Despite having an additional iodine atom rela-
tive to FTY720, BZM055 demonstrates very similar properties,
both in terms of the biological activity of its phosphate (affinity
for S1P receptors, activity in the lymphocyte depletion assay)
and pharmacokinetics properties of both the parent and phos-
phorylated analogue. Imaging studies with [123I]BZM055 are
ongoing, and the results will be reported in due course.
In contrast, and despite its resemblance to FTY720 and
BZM055, compound 2b is phosphorylated to a form that pen-
etrates the brain to a much smaller extent. This property
makes it unsuitable as a radiotracer to mimic the distribution
of FTY720 in vivo. Nevertheless, 2b remains an interesting tool,
as it might allow study of the importance of central S1P recep-
tor modulation on the pathogenesis of MS, for instance by
comparing its efficacy in animal models of the disease to
BZM055.
Preparative HPLC
Method C: Gilson Trilution LC, column: SunFire C18 5 mm, 30ꢃ
100 mm, eluent: H2O (+0.1% TFA)/CH3CN (+0.1% TFA) from 85:15
to 65:35 in 16 min; flow rate: 50 mLminꢀ1
.
Compounds
2-Amino-2-[2-(2-iodo-4-octylphenyl)ethyl]-1,3-propandiol
and 2-amino-2-[2-(3-iodo-4-octylphenyl)ethyl]-1,3-propandiol
(2a)
(2b): Ag2SO4 (5.07 g, 16.3 mmol) and I2 (4.13 g, 16.3 mmol) were
added to a stirred solution of FTY720 (5.0 g, 16.3 mmol) in CH2Cl2
(150 mL) and CH3OH (7.5 mL) at RT, which was then treated with
CF3SO3Ag (0.21 g, 0.8 mmol), and stirred for 18 h at 308C. The
yellow solid AgI was filtered off, the organic phase washed with
15% aqueous NaHCO3, dried over Na2SO4, filtered and evaporated
to dryness. The residue was purified using SFC to yield, after evap-
oration, 2a (2.1 g, 60%) and 2b (2.1 g, 60%) as white solids: Thar
SFC 200, Chiralpak IC, 30ꢃ250 mm, isocratic: CO2/2-propanol/2-
propylamine 75:25:0.25; flow rate: 90 gminꢀ1; back pressure: 15ꢃ
106 Pa.
2a: SFC tR =5.58 min; mp: 848C; 1H NMR (600 MHz, [D6]DMSO):
d=7.61 (s, 1H), 7.19–7.06 (m, 2H), 4.54 (brs, 2H), 3.17–3.27 (m,
4H), 2.58–2.64 (m, 2H), 2.41–2.47 (m, 2H), 2.06 (brs, 2H), 1.39–1.42
(m, 4H), 1.20–1.31 (m, 10H), 0.85 ppm (t, J=6.8 Hz, 3H); 13C NMR
(600 MHz, [D6]DMSO): d=142.6, 142.1, 138.5, 129.0, 128.6, 100.5,
65.1, 55.7, 35.4, 33.9, 33.6, 31.2, 30.8, 28.7, 28.6, 28.5, 22.0,
˜
13.9 ppm; IR (solid film): n=3373, 3344, 3235, 3055, 2953, 2920,
2851, 1578, 1463, 1454, 1060, 1045, 1035, 983 cmꢀ1
; UPLC–
MS[SQD] (ES+): m/z 434 [M+H]; FTMS-pESI m/z [M+H]+; Anal.
Experimental Section
Chemistry
calcd for C19H33O2NI: 434.15505, found: 434.15522.
All chemicals, reagents, and solvents for the synthesis of the com-
pounds were analytical grade, purchased from commercial sources
and used without purification, unless otherwise specified. H NMR
spectra were acquired on Bruker (360 MHz), Varian Mercury
(400 MHz), or Bruker Advance (600 MHz) instruments. Chemical
shift (d) values are reported in parts per million (ppm) relative to
the residual solvent peak. IR spectra were measured in transmis-
sion as a solid film on a Bruker Tensor 27 FTIR spectrometer cou-
2b: SFC tR =4.82 min; mp: 638C; 1H NMR (600 MHz, [D6]DMSO):
d=7.63 (s, 1H), 7.09–7.27 (m, 2H), 4.50 (brs, 2H), 3.10–3.29 (m,
4H), 2.59 (t, J=7.5 Hz, 2H), 2.55–2.36 (m, 2H), 1.65 (brs, 2H), 1.39–
1.55 (m, 4H), 1.16–1.35 (m, 10H), 0.74–0.93 ppm (m, 3H); 13C NMR
(600 MHz, [D6]DMSO): d=143.3, 141.5, 138.4, 129.2, 128.4, 100.5,
65.1, 55.6, 39.0, 36.5, 31.2, 29.9, 28.7, 28.7, 28.6, 28.4, 27.8, 22.1,
1
˜
13.9 ppm; IR (solid film): n=3346, 3283, 3043, 2951, 2920, 2853,
2705, 1576, 1464, 1456, 1049, 1022, 961, 828 cmꢀ1; UPLC–MS[SQD]
ChemMedChem 2011, 6, 667 – 677
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671

E. Briard et al.
MED
(ES+): m/z 434 [M+H]; FTMS-pESI m/z [M+H]+; Anal. calcd for
C19H33O2NI: 434.15505, found: 434.15519.
(1.04 mL, 3.46 mmol) were added to a solution of 3a (530 mg,
1.15 mmol) at 08C in CH2Cl2 (4 mL) and THF (4 mL). After 18 h at
RT, 30% aqueous H2O2 (0.35 mL, 11.54 mmol) was added dropwise,
and the mixture stirred at RT for an additional 90 min. The reaction
mixture was quenched with saturated aqueous Na2S2O3, and the
aqueous phase extracted with CH2Cl2. The combined organic frac-
tions were dried over Na2SO4, filtered and concentrated. The crude
product was purified by flash chromatography on silica gel (MTBE,
100%) to give (R/S)-4a as a yellow oil (295 mg, 39%). Both enan-
tiomers were isolated in optically pure form after preparative enan-
tioselective HPLC (Chiralpak AS-PREP, heptane/EtOH/CH3OH, 90:5:5,
1 mLminꢀ1, l 210 nm).
2-Amino-2-[2-[3-iodo-4-(heptyloxy)phenyl]ethyl]-1,3-propanediol
(2c): Compound 2c was obtained as a white solid (2.7 g, 64%)
from 1 according to the procedure described for the synthesis of
2a and 2b by using wet CH2Cl2 as solvent. Purification of the resi-
due by flash chromatography (CH2Cl2/CH3OH/NH4OH, 90:10:1)
gave 2c as white solid (2.7 g, 64%): tRA =2.62 min; mp: 718C;
1H NMR (600 MHz, [D6]DMSO): d=7.57 (d, J=6.7 Hz, 1H), 7.13 (dd,
J=8.4, 1.7 Hz, 1H), 6.87 (d, J=8.5 Hz, 1H), 4.47 (brs, 2H), 3.96 (t,
J=6.3 Hz, 2H), 3.27–3.07 (m, 4H), 2.49–2.42 (m, 2H), 1.61–1.76 (m,
2H), 1.49–1.39 (brs, 2H), 1.36–1.18 (m, 10H), 0.86 ppm (t, J=
6.8 Hz, 3H); 13C NMR (600 MHz, [D6]DMSO): d=155.0, 138.3, 137.6,
129.3, 112.4, 86.5, 68.5, 65.2, 55.5, 36.8, 31.3, 28.3, 25.5, 22.0,
(S)-4a (110.7 mg): tR =5.24 min; ee>99%; [a]2D0 =ꢀ5.67 (c=0.14 in
CH3Cl3); 1H NMR (600 MHz, [D6]DMSO): d=7.97 (s, 1H), 7.64 (s,
1H), 7.28–7.14 (m, 2H), 4.30–4.12 (m, 2H), 3.83 (d, J=5.05 Hz, 2H),
2.76–2.56 (m, 2H), 2.50–2.38 (m, 2H), 1.81–1.61 (m, 2H), 1.61–1.42
(m, 2H), 1.42–1.34 (m, 18H), 1.34–1.17 (m, 10H), 0.85 ppm (t, J=
6.96 Hz, 3H); 13C NMR (600 MHz, [D6]DMSO): d=157.97, 142.69,
140.70, 138.68, 129.13, 128.74, 100.38, 82.02, 81.97, 69.60, 69.10,
59.35, 35.66, 33.87, 33.34, 31.26, 30.79, 29.42, 28.74, 28.63, 28.54,
˜
13.4 ppm; IR (solid film): n=3351, 3322, 3289, 3123, 2923, 2856,
1598, 1492, 1471, 1280, 1250, 1078, 1039, 979, 811 cmꢀ1; UPLC–
MS[SQD] (ES+): m/z 435 [M]; FTMS-pESI m/z [M+H]+; Anal. calcd
for C18H30O3NI: 435.12703, found: 435.12698.
4-Hydroxymethyl-4-[2-(2-iodo-4-octylphenyl)ethyl]oxazolidin-2-
one (3a): Benzyl chloroformate (0.25 mL, 1.7 mmol) was added to a
suspension of 2b (0.68 g, 1.6 mmol) in 2n NaOH (10 mL), the mix-
ture was stirred at RT overnight, then acidified with 1n HCl and ex-
tracted with CH2Cl2. The combined organic phases were dried over
Na2SO4, filtered and concentrated to give 3a as a white powder
(540 mg, 75%): tRA =3.77 min; 1H NMR (600 MHz, [D6]DMSO): d=
7.72 (s, 1H), 7.63 (s, 1H), 7.28–7.11 (m, 2H) 5.14 (t, J=5.55 Hz, 1H)
4.19 (d, J=8.68 Hz, 1H) 4.09 (d, J=8.48 Hz, 1H) 3.42–3.34 (m, 2H)
2.70–2.56 (m, 2H) 2.50–2.45 (m, 2H) 1.64 (t, J=8.68 Hz, 2H) 1.55–
1.43 (m, 2H) 1.32–1.15 (m, 10H) 0.85 ppm (t, J=6.96 Hz, 3H);
13C NMR (600 MHz, [D6]DMSO): d=158.4, 142.6, 141.1, 138.6, 129.2,
128.7, 100.4, 70.0, 65.2, 60.5, 36.1, 33.9, 31.2, 28.6, 22.1, 14.0 ppm;
˜
22.08, 13.97 ppm; IR (solid film): n=3244, 2979, 2955, 2926, 2855,
1759, 1483, 1457, 1396, 1371, 1261, 1172, 1040, 1004 cmꢀ1; UPLC–
MS[SQD] (ES+): m/z 696 [M+HCOOꢀ].
Phosphoric acid di-tert-butyl ester (S)-4-[2-(3-iodo-4-octylphen-
yl)ethyl]-2-oxo-oxazolidin-4-ylmethyl ester ((S)-4b): 1H-tetrazole
(621 mg, 8.87 mmol) and di-tert-butyl diethylphosphoramidite
(1.59 mL, 5.32 mmol) were added to a solution of 3b (815 mg,
1.77 mmol) at 08C in CH2Cl2 (5 mL) and THF (5 mL). After 18 h at
RT, 30% aqueous H2O2 (0.54 mL, 17.7 mmol) was added dropwise,
and the mixture was stirred at RT for an additional 90 min. The re-
action mixture was quenched with saturated aqueous Na2S2O3, and
the aqueous phase extracted with CH2Cl2. The combined organic
fractions were dried over Na2SO4, filtered and concentrated. The
crude product was purified by flash chromatography on silica gel
(CH2Cl2/CH3OH, 90:10) to give (R/S)-4b as a yellow oil (440 mg,
38%). Both enantiomers were isolated in optically pure form after
preparative enantioselective HPLC (Chiralpak AS-PREP, heptane/
EtOH/CH3OH, 80:10:10, 1 mLminꢀ1, l 210 nm).
˜
IR (solid film): n=3318, 3248, 3051, 3028, 2995, 2953, 2926, 2886,
2851, 1769, 1598, 1459, 1407, 1274, 1250, 1052 cmꢀ1; UPLC–
MS[SQD] (ES+): m/z 460 [M+H].
4-Hydroxymethyl-4-[2-(3-iodo-4-octylphenyl)ethyl]oxazolidin-2-
one (3b) (815 mg, 77%): Prepared in the same way as compound
1
3a: tRA =3.79 min; H NMR (600 MHz, [D6]DMSO): d=7.65 (s, 1H),
7.59 (s, 1H), 7.19–7.10 (m, 2H), 5.08 (t, J=5.49 Hz, 1H), 4.16 (d, J=
8.46 Hz, 1H), 4.06 (d, J=8.59 Hz, 1H), 3.41–3.30 (m, 2H), 2.69–2.55
(m, 2H), 2.47–2.42 (m, 2H), 1.63 (dd, J=9.66, 7,64 Hz, 2H), 1.52–
1.47 (m., 2H), 1.25–1.18 (m, 10H), 0.91–0.74 ppm (m, 3H); 13C NMR
(600 MHz, [D6]DMSO): d=158.4, 142.0, 141.7, 138.5, 1293, 128.5,
100.6, 69.9, 65.3, 60.6, 37.0, 31.2, 29.9, 28.75, 28.7, 27.7, 22.1,
(S)-4b (203 mg): tR =11.76 min; ee>98.6%; [a]2D0 =ꢀ4.67 (c=0.15
1
in CH3Cl3); H NMR (600 MHz, [D6]DMSO): d=7.88 (s, 1H), 7.68 (s,
1H), 7.23–7.07 (m, 2H), 4.23–4.04 (m, 2H), 3.80–3.61 (m, 2H), 1.88–
1.65 (m, 2H), 1.50–1.46 (m, 2H), 1.42–1.40 (m, 18H), 1.37–1.17 (m,
14H), 0.85 ppm (t, J=6.96 Hz, 3H); 13C NMR (600 MHz, [D6]DMSO):
d=158.03, 142.11, 141.32, 138.54, 129.37, 128.45, 100.67, 81.98,
81.93, 69.48, 69.26, 59.47, 40.02, 36.60, 31.25, 29.92, 29.41, 29.38,
˜
13.9 ppm; IR (solid film): n=3253, 3045, 3012, 2952, 2934, 2917,
851, 1721, 1471, 1409, 1042, 1019 cmꢀ1; UPLC–MS[SQD] (ES+): m/z
460 [M+H].
˜
28.77, 28.66, 28.63, 27.44, 22.09, 13.98 ppm; IR (solid film): n=
3155, 2984, 2956, 2923, 2856, 1770, 1738, 1409, 1393, 1369, 1243,
1043, 1003 cmꢀ1; UPLC–MS[SQD] (ES+): m/z 696 [M+HCOOꢀ].
4-[2-(4-Heptyloxy-3-iodophenyl)ethyl]-4-hydroxymethyloxazoli-
din-2-one (3c) (660 mg, 78%): Prepared in the same way as 3a:
t
RA =3.37 min; 1H NMR (600 MHz, [D6]DMSO): d=7.71–7.54 (m,
Phosphoric acid di-tert-butyl ester (S)-4-[2-(4-heptyloxy-3-iodo-
phenyl)ethyl]-2-oxo-oxazolidin-4-ylmethyl ester ((S)-4c): 1H-tetra-
zole (501 mg, 7.15 mmol) and di-tert-butyl diethylphosphoramidite
(1.28 mL, 4.29 mmol) were added to a solution of 3c (660 mg,
1.43 mmol) at 08C in CH2Cl2 (5 mL) and THF (5 mL). After 18 h at
RT, 30% aqueous H2O2 (0.44 mL, 14.3 mmol) was added dropwise
and the mixture was stirred at RT for an additional 90 min. The re-
action mixture was quenched with saturated aqueous Na2S2O3, and
the aqueous phase extracted with CH2Cl2. The combined organic
fractions were dried over Na2SO4, filtered and concentrated. The
crude product was purified by flash chromatography on silica gel
(CH2Cl2/CH3OH, 90:10) to give (R/S)-4c as a yellow oil (580 mg,
62%). Both enantiomers were isolated in optically pure form after
2H), 7.15 (dd, J=8.39, 1.68 Hz, 1H), 6.88 (d, J=8.39 Hz, 1H), 5.09
(t, J=5.57 Hz, 1H), 4.22–4.08 (m, 1H), 4.04 (d, J=8.70 Hz, 1H), 3.96
(t, J=6.26 Hz, 2H), 3.41–3.24 (m, 2H), 2.58–2.35 (m, 2H), 1.77–1.56
(m, 4H), 1.56–1.38 (m, 2H), 1.38–1.14 (m, 6H), 0.86 (t, J=6.71 Hz,
3H); 13C NMR (600 MHz, [D6]DMSO): d=158.4, 155.3, 138.3, 136.0,
129.4, 112.4, 86.6, 69.9, 68.5, 65.3, 60.6, 37.2, 31.2, 28.7, 28.3, 28.0,
˜
27.4, 25.5, 22.0, 14.0 ppm; IR (solid film): n=3422, 3314, 3028,
2930, 2870, 2857, 1727, 1599, 1493, 1400, 1282, 1260, 1043,
944 cmꢀ1; UPLC–MS[SQD] (ES+): m/z 462 [M+H].
Phosphoric acid di-tert-butyl ester (S)-4-[2-(2-iodo-4-octylphenyl)-
ethyl]-2-oxo-oxazolidin-4-ylmethyl ester ((S)-4a): 1H-tetrazole
(404 mg, 5.77 mmol) and di-tert-butyl diethylphosphoramidite
672
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ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 667 – 677

BZM055
preparative enantioselective HPLC (Chiralpak AS-PREP, hexane/
EtOH/CH3OH, 70:15:15+0.1% DEA, 1 mLminꢀ1, l 220 nm).
13C NMR (600 MHz, [D6]DMSO, CH3COOH 5%): d=155.51, 138.37,
135.39, 129.39, 112.37, 86.57, 68.55, 65.02, 60.95, 59.03, 33.45,
˜
31.21, 28.29, 25.49, 21.98, 13.77 ppm; IR (solid film): n=3214, 2926,
(S)-4c (180 mg): tR =8.19 min; ee>99.9%; [a]2D0 =+4.68 (c=0.17 in
CH3Cl3); H NMR (600 MHz, [D6]DMSO): d=7.88 (s, 1H), 7.62 (d, J=
2856, 1730, 1626, 1600, 1536, 1513, 1491.1468, 1281, 1251, 1155,
1074, 944, 810 cmꢀ1; UPLC–MS[SQD] (ES+): m/z 516 [M+H]; FTMS-
pESI m/z [M+H]+; Anal. calcd for C18H32O6NIP: 516.10065, found:
516.10087.
1
1.82 Hz, 1H), 7.17 (dd, J=8.28, 1.82 Hz, 1H), 6.90 (d, J=8.28 Hz,
1H), 4.17–4.11 (m, 2H), 3.97 (t, J=6.16 Hz, 2H), 3.78 (d, J=4.84 Hz,
2H), 2.56–2.44 (m, 2H), 1.78–1.63 (m, 4H), 1.47–1.37 (m, 20H),
1.35–1.23 (m, 6H), 0.86 ppm (t, J=6.86 Hz, 3H); 13C NMR
(600 MHz, [D6]DMSO): d=158.03, 155.40, 138.38, 135.53, 129.39,
112.44, 86.70, 81.97, 81.92, 69.51, 69.27, 68.55, 59.44, 36.87, 31.27,
29.41, 28.57, 23.33, 27.15, 25.53, 22.05, 13.98 ppm; IR (solid film):
(E)-6-Iodohex-5-en-1-ol (6a): In a flame-dried flask, LiEt3BH (1m in
THF, 7.7 mL, 1.1 equiv) was added to a solution of Cp2ZrCl2 (2.27 g,
1.1 equiv) in THF (20 mL). The resulting mixture was stirred for
30 min at RT, then treated with a solution of tert-butylhex-5-ynyloxy-
dimethylsilane (1.5 g, 7.01 mmol) in THF (10 mL). The mixture
turned into a clear yellow solution within a few seconds after the
end of the addition. It was stirred for 30 min at RT prior to the ad-
dition of a solution of N-iodosuccinimide (1.75 g, 1.1 equiv) in THF,
then stirred for an additional 3 h at RT. The mixture was poured
into saturated NaHCO3, and the aqueous phase was extracted with
EtOAc. The organic phase was washed with 1m Na2S2O3 and brine,
dried over Na2SO4 and concentrated in vacuo to afford a crude
yellow liquid (2.1 g) which was purified by flash chromatography
using cHex/EtOAc (100:0!95:5). This yielded tert-butyl-((E)-6-iodo-
hex-5-enyloxy)dimethylsilane as a colorless oil (1.45 g) with 70%
purity. This oil was solubilized in Et2O/CH3OH (20 mL, 1:1), and
CH3COCl (1.5 mL) was added dropwise at 08C. The mixture was
stirred at RT for 61 h. The reaction was quenched with TEA
(2.85 mL). The salts were removed by filtration and washed with
Et2O. The filtrate was washed with saturated NH4Cl, dried over
Na2SO4, and the solvent was removed in vacuo to give a crude
brown oil (1.0 g) which was purified by flash chromatography on
silica gel using cHex/EtOAc (100:0!50:50). From the purification,
440 mg of light-brown oil was collected containing 6a plus 10%
hexen-1-ol and 5% hexyn-1-ol. The volatile byproducts were re-
moved under high vacuum to afford pure 6a as a light-brown oil
(330 mg, 58%): Rf =0.28 (cHex/EtOAc); 1H NMR (400 MHz, CDCl3)
d=6.50 (m, 1H), 6.0 (d, J=7.1 Hz, 1H), 3.63 (t, J=7.1 Hz, 2H), 2.09
(m, 2H), 1.7–1.3 ppm (m, 4H).
˜
n=3159, 2980, 2953, 2928, 2871, 2857, 1769, 1738, 1599, 1490,
1469, 1406, 1398, 1369, 1281, 1252, 1241, 1043, 1002, 875, 813;
UPLC–MS[SQD] (ES+): m/z 698 [M+HCOOꢀ].
Phosphoric acid mono-[(S)-2-amino-2-hydroxymethyl-4-(2-iodo-
4-octylphenyl)butyl] ester ((S)-5a): 10% aqueous LiOH (0.5 mL,
2.09 mmol) was added to a solution of (S)-4a (20 mg, 0.031 mmol)
in EtOH (0.5 mL). After 20 h at 608C, the reaction mixture was
cooled to RT and stirred for an additional two days. Concentrated
aqueous HCl (0.5 mL) was then added, and the solution was stirred
at RT for 1 h, neutralized with 4n aqueous NaOH, and concentrat-
ed. The residue was taken up in CH2Cl2 (2 mL), filtered on Hyflo,
and the filtrate washed twice with CH2Cl2. The solution was con-
centrated to give (S)-5a as a white powder (5 mg, 32%); tR =
135.8 min; ee>92.3% (Sumichiral OA-7000, KH2PO4 (20 mm
1
pH 3.0)/CH3CN, 30:70, 1.5 mLminꢀ1, l 228 nm); H NMR (600 MHz,
[D6]DMSO, CF3COOH 5%): d=8.08 (brs, 3H), 7.63 (s, 1H), 7.24–7.11
(m, 2H), 4.03–4.99 (m, 2H), 3.66–3.54 (m, 2H), 2.71–2.59 (m, 2H),
2.51–2.36 (m, 2H), 1.77 (dd, J=10.19, 7.16 Hz, 2H), 1.52–1.48 (m,
2H), 1.29–1.10 (m, 10H), 0.83 ppm (t, J=6.86 Hz, 3H); 13C NMR
(600 MHz, [D6]DMSO, CF3COOH 5%): d=142.84, 140.66, 138.69,
129.09, 128.72, 100.13, 62.01, 61.0, 58.80, 33.88, 32.88, 32.12, 32.21,
˜
30.79, 28.69, 28.55, 22.00, 13.77 ppm; IR (zwitterion, solid film): n=
3346, 3114, 3043, 2956, 2924, 2854, 2653, 2578, 1623, 1600, 1539,
1452, 1271, 1160, 1096, 1059, 1040, 834, 815 cmꢀ1; UPLC–MS[SQD]
(ES+): m/z 514 [M+H]; FTMS-pESI m/z [M+H]+; Anal. calcd for
C19H34O5NIP: 514.12138, found: 514.12167. (S)-5b and (S)-5c were
obtained in a similar manner.
(4-{2-[4-((E)-6-Iodohex-5-enyloxy)phenyl]ethyl}-2-methyl-4,5-di-
hydro-oxazol-4-yl)methanol (8a): DIAD (0.215 mL, 1.0 equiv) was
added at 08C to 7 (260 mg, 1.1 mmol), 6a (250 mg, 1.0 equiv), and
PPh3 (290 mg, 1.0 equiv) in THF (10 mL). The reaction mixture was
stirred for 18 h at RT and 24 h at 508C, then treated with 0.8 equiv
DIAD and PPh3. The mixture was stirred for an additional 72 h at
508C, then partitioned between EtOAc and aqueous saturated
NH4Cl. The organic phase was separated, dried over Na2SO4, and
concentrated in vacuo to afford a crude beige oil (1.54 g), which
was purified by flash chromatography on silica gel using CH2Cl2/
CH3OH (100:0!90:10) as eluent. Compound 8a was isolated as a
colorless oil (290 mg, 59% yield): Rf =0.45 (CH2Cl2/CH3OH, 95:5);
1H NMR (400 MHz, CDCl3) d=7.08 (d, J=8.6 Hz, 2H), 6.79 (d, J=
8.6 Hz, 2H), 6.52 (m, 1H), 6.01 (d, J=14 Hz, 1H), 4.18 (AB, J=73,
8.5 Hz, 2H), 3.92 (t, J=6.3 Hz, 2H), 3.58 (AB, J=107, 11.4 Hz, 2H),
2.54 (m, 2H), 2.12 (m, 2H), 2.06 (s, 3H), 1.95–1.50 ppm (m, 6H);
LC–MS: tRB =4.84 min; m/z: 444.0 [M+H].
Phosphoric acid mono-[(S)-2-amino-2-hydroxymethyl-4-(3-iodo-
4-octylphenyl)butyl] ester ((S)-5b): 15 mg, 63%; tR =22.09 min;
ee>90% (Sumichiral OA-7000, KH2PO4 (5 mm pH 3.0)/CH3CN,
,
50:50, 1.0 mLminꢀ1 l 224 nm); 1H NMR (600 MHz, [D6]DMSO,
CF3COOH 5%): d=8.05 (brs, 3H), 7.68 (s, 1H), 7.23–7.08 (m, 2H),
3.95 (d, J=4.84 Hz, 2H), 3.57–3.53 (m, 2H), 2.60 (t, J=7.57 Hz, 2H),
2.57–2.51 (m, 2H), 1.85–1.83 (m, 2H), 1.49–1.45 (m, 2H), 1.15–1.35
(m, 10H), 0.84 ppm (t, J=6.56 Hz, 3H); 13C NMR (600 MHz,
[D6]DMSO, CF3COOH 5%): d=142.31, 141.13, 138.52, 129.35,
128.43, 100.54, 65.00, 60.95, 59.02, 33.16, 31.21, 29.92, 28.71, 28.65,
˜
28.59, 29.96, 22.02, 13.79 ppm; IR (zwitterion, solid film): n=3154,
3042, 2954, 2924, 2853, 2574, 1732, 1623, 1599, 1537, 1465, 1158,
1057, 1043, 814 cmꢀ1; UPLC–MS[SQD] (ES+): m/z 514 [M+H];
FTMS-pESI m/z [M+H]+; Anal. calcd for C19H34O5NIP: 514.12138,
found: 514.12150.
Phosphoric acid mono-[(S)-2-amino-4-(4-heptyloxy-3-iodophen-
yl)-2-hydroxymethylbutyl] ester ((S)-5c): 9 mg, 57%; tR =32.6 min;
ee>98.0% (Sumichiral OA-7000, KH2PO4 (20 mm pH 3.0)/CH3CN,
(4-{2-[4-((Z)-6-Iodohex-5-enyloxy)phenyl]ethyl}-2-methyl-4,5-di-
hydro-oxazol-4-yl)methanol (8b) and (4-{2-[4-(5-iodohex-5-en-
yloxy)phenyl]ethyl}-2-methyl-4,5-dihydro-oxazol-4-yl)methanol
(8c): Compounds 8b and 8c were prepared from 7 and 6b
(250 mg, 1.0 equiv) or 6c (728 mg, 1.5 equiv) according to the pro-
cedure described for the synthesis of 8a. Compounds 8b (403 mg,
53.5% yield) and 8c (520 mg, 54.6% yield) were isolated as clear
oils.
40:60, 1.5 mLminꢀ1
,
l 228 nm); 1H NMR (600 MHz, [D6]DMSO,
CH3COOH 5%): d=8.04 (brs, 3H), 7.62 (s, 1H), 7.15 (d, J=6.86 Hz,
1H), 6.89 (d, J=8.48 Hz, 1H), 3.99–3.93 (m, 4H), 3.55 (brs, 2H),
2.53–2.51 (m, 2H), 1.70 (d, J=11.50 Hz, 2H), 1.73–1.64 (m, 2H),
1.50–1.33 (m, 2H), 1.35–1.17 (m, 6H), 0.85 ppm (t, J=6.76 Hz, 3H);
ChemMedChem 2011, 6, 667 – 677
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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673

E. Briard et al.
MED
1
8b: Rf =0.28 (CH2Cl2/CH3OH, 95:5); H NMR (400 MHz, CDCl3) d=
7.08 (d, J=8.6 Hz, 2H), 6.81 (d, J=8.6 Hz, 2H), 6.25–6.15 (m, 2H),
4.21 (AB, J=74, 8.5 Hz, 2H), 3.94 (t, J=6.3 Hz, 2H), 3.60 (AB, J=
111, 11.4 Hz, 2H), 2.54 (m, 2H), 2.22 (m, 2H), 2.10 (s, 3H), 1.95–
1.55 ppm (m, 6H); LC–MS: tRB =4.76 min, m/z: 443.9 [M+H].
tion of 8a (230 mg, 0.52 mmol) at 08C in 4 mL CH2Cl2/THF (1:1),
then stirred at RT for 6 h. H2O2 (30% wt.% in H2O, 0.159 mL,
10 equiv) was added, and the mixture was stirred for 1.5 h at RT.
The reaction mixture was quenched by careful addition of a solu-
tion of 1n Na2S2O3 (10 mL), and the aqueous phase was extracted
with CH2Cl2. The organic phases were combined, washed with
brine, dried over Na2SO4, and concentrated in vacuo to afford a
crude oil (500 mg), which was purified by flash chromatography on
silica gel (eluent: CH2Cl2/CH3OH 100:0!90:10). Compound 10a
(107 mg) was isolated as clear oil with a purity of ~50% and used
as such in the next step. LC–MS: tRB =5.53 min, m/z: 636.1 [M+H].
8c: Rf =0.31 (CH2Cl2/CH3OH, 95:5); 1H NMR (400 MHz, CDCl3) d=
7.08 (d, J=8.6 Hz, 2H), 6.81 (d, J=8.6 Hz, 2H), 6.04 (m, 1H), 5.71
(m, 1H), 4.18 (AB, J=73, 8.5 Hz, 2H), 3.94 (t, J=6.3 Hz, 2H), 3.58
(AB, J=107, 11.4 Hz, 2H), 2.54 (m, 2H), 2.45 (m, 2H), 2.06 (s, 3H),
1.95–1.65 ppm (m, 6H); LC–MS: tRB =4.76 min, m/z: 444.0 [M+H].
2-Amino-2-{2-[4-((E)-6-iodohex-5-enyloxy)phenyl]ethyl}propane-
1,3-diol·HCl (9a·HCl): Aqueous HCl (33%, 2.05 mL) was added to a
solution of 8a (60 mg, 0.135 mmol) in EtOH (2 mL), and the reac-
tion mixture was stirred at 858C for 2.5 h. The solvents were re-
moved in vacuo to afford a beige paste. After trituration in Et2O,
(R/S)-phosphoric acid di-tert-butyl ester 4-{2-[4-((Z)-6-iodohex-5-
enyloxy)phenyl]ethyl}-2-methyl-4,5-dihydro-oxazol-4-ylmethyl
ester (10b) and (R/S)-phosphoric acid di-tert-butyl ester 4-{2-[4-
(5-iodohex-5-enyloxy)phenyl]ethyl}-2-methyl-4,5-dihydro-oxazol-
4-ylmethyl ester (10c): Compounds 10b and 10c were prepared
from 8b (330 mg, 0.744 mmol) or 8c (200 mg, 0.45 mmol) accord-
ing to the procedure described for the synthesis of 10a. Com-
pounds 10b (665 mg, 98% yield, 70% purity) and 10c (230 mg,
80% yield, 70% purity) were used as such in the next step.
1
9a·HCl was isolated as a beige powder (48 mg, 78% yield). H NMR
(600 MHz, [D6]DMSO): d=7.87 (brs, 3H), 7.10 (d, J=8.7 Hz, 2H),
6.84 (d, J=8.7 Hz, 2H), 6.53 (dt, J=14.4 and 7.2 Hz, 1H), 6.24 (d,
J=14.4 Hz, 1H), 5.39 (t, J=5.2 Hz, 2H), 3.91 (t, J=6.5 Hz, 2H), 3.50
(m, 4H), 2.53 (m, 2H), 2.09 (m, 2H), 1.75 (m, 2H), 1.67 (m, 2H),
1.48 ppm (q, J=8.3 Hz, 2H); 13C NMR (150 MHz, [D6]DMSO): d=
24.3, 27.4, 28.0, 33.4, 35.0, 60.2, 60.9, 77.0, 114.3, 129.1, 133.4,
(R/S)-phosphoric acid mono-{2-amino-2-hydroxymethyl-4-[4-((E)-
6-iodohex-5-enyloxy)phenyl]butyl} ester (11a): 10a (107 mg,
0.168 mmol) in EtOH (2.5 mL) was treated with 33% aqueous HCl
(2.56 mL) and stirred at 858C for 2.5 h, then concentrated in vacuo
to afford a beige paste. After trituration in Et2O, 11 a was isolated
˜
146.1, 156.8 ppm; IR (solid film): n=3365, 3278, 2938, 2866, 1611,
1582, 1513, 1246, 1179, 1069, 1046, 1027, 949, 829 cmꢀ1; LC–MS:
t
RB =4.62 min; m/z: 419.9 [M+H]; HRMS-pESI m/z [M+H]+ calcd for
C17H27O3NI: 420.10301, found: 420.10287.
as a
beige powder (71 mg, 84% yield). 1H NMR (600 MHz,
[D6]DMSO): d=8.27 (brs, 3H), 7.10 (d, J=7.5 Hz, 2H), 6.81 (d, J=
8.1 Hz, 2H), 6.53 (dt, J=14.4 and 7.0 Hz, 1H), 6.23 (d, J=14.4 Hz,
1H), 3.9 (m, 4H), 3.4–3.6 (m, 2H), 2.52 (m, 2H), 2.09 (q, J=7.1 Hz,
2H), 1.77 (m, 2H), 1.66 (m, 2H), 1.48 ppm (m, 2H); 13C NMR
(150 MHz, [D6]DMSO): d=24.3, 27.4, 28.0, 33.7, 35.0, 59.3, 60.4,
64.2, 67.0, 77.0, 114.3, 129.1, 133.3, 146.1, 156.8 ppm; IR (solid film):
2-Amino-2-{2-[4-((Z)-6-iodohex-5-enyloxy)phenyl]ethyl}propane-
1,3-diol·TFA (9b·TFA): 9b·TFA was prepared from 8b (75 mg,
0.169 mmol) according to the procedure described for the synthe-
sis of 9a. After an additional purification by reverse preparative
HPLC (method C), 9b·TFA was obtained as a white powder (30 mg,
1
33% yield). H NMR (600 MHz, [D6]DMSO): d=7.77 (brs, 3H), 7.09
˜
n=2941, 2868, 1620, 1612, 1583, 1540, 1512, 1246, 1180, 1044,
(d, J=8.7 Hz, 2H), 6.84 (d, J=8.5 Hz, 2H), 6.40 (d, J=7.3 Hz, 1H),
6.30 (q, J=7.1 Hz, 1H), 5.39 (t, J=5.0 Hz, 2H), 3.93 (t, J=6.5 Hz,
2H), 3.50 (m, 4H), 2.52 (m, 2H), 2.14 (m, 2H), 1.72 (m, 4H),
1.53 ppm (q, J=7.4 Hz, 2H); 13C NMR (150 MHz, [D6]DMSO): d=
24.0, 27.4, 28.1, 33.4, 34.0, 60.1, 61.0, 67.1, 84.6, 114.3, 129.1, 133.3,
1016, 952, 836 cmꢀ1; LC–MS: tRB =4.53 min; m/z: 500.0 [M+H];
HRMS-pESI m/z [M+H]+ calcd for C17H28O6NIP: 500.06935, found:
500.06949.
(R/S)-phosphoric acid mono-{2-amino-2-hydroxymethyl-4-[4-((Z)-
6-iodohex-5-enyloxy)phenyl]butyl} ester (11b) and (R/S)-phos-
phoric acid mono-{2-amino-2-hydroxymethyl-4-[4-(5-iodohex-5-
enyloxy)phenyl]butyl} ester (11c): Compounds 11 b and 11 c were
prepared from 10b (330 mg, 0.744 mmol, 70% purity) or 10c
(230 mg, 0.362 mmol, 70% purity) according to the procedure de-
scribed for the synthesis of 11 a, except that for 11 b, the reaction
was performed in dioxane at 508C for 4 h. 11 b was isolated as a
white powder (152 mg, 42% yield), and after purification by prepa-
rative reversed-phase HPLC (method C), 11 c was isolated as a
beige powder (54 mg, 30% yield).
˜
140.9, 156.8 ppm; IR (solid film): n=3404, 2983, 2934, 2866, 1596,
1613, 1514, 1246, 1185, 1073, 10489, 837 cmꢀ1; LC–MS: tRB
=
4.42 min; m/z: 420.0 [M+H]; HRMS-pESI m/z [M+H]+ calcd for
C17H27O3NI: 420.10301, found: 420.10294.
2-Amino-2-{2-[4-(5-iodohex-5-enyloxy)phenyl]ethyl}propane-1,3-
diol·HCl (9c·HCl): 9c·HCl was prepared from 8c (63 mg,
0.142 mmol) according to the procedure described for the synthe-
sis of 9a, except that the reaction was performed in dioxane in-
stead of EtOH at 508C for 20 h and then 708C for 4 h. 9c·HCl was
obtained as
a
beige powder (35 mg, 54% yield). 1H NMR
1
(600 MHz, [D6]DMSO): d=7.87 (brs, 3H), 7.10 (d, J=8.5 Hz, 2H),
6.84 (d, J=8.5 Hz, 2H), 6.19 (s, 1H), 5.72 (s, 1H), 5.39 (t, J=5.0 Hz,
2H), 3.93 (t, J=6.5 Hz, 2H), 3.51 (m, 4H), 2.53 (m, 2H), 2.43 (t, J=
7.1 Hz, 2H), 1.74 (m, 2H), 1.68 (m, 2H), 1.58 ppm (m, 2H); 13C NMR
(150 MHz, [D6]DMSO): d=25.2, 27.2, 27.4, 33.4, 44.0, 60.2, 60.9,
11 b: H NMR (600 MHz, [D6]DMSO): d=8.43 (brs, 3H), 7.10 (d, J=
7.5 Hz, 2H), 6.81 (d, J=7.7 Hz, 2H), 6.40 (d, J=7.7 Hz, 1H), 6.30 (q,
J=6.9 Hz, 1H), 3.8–4.0 (m, 4H), 3.4–3.7 (m, 2H), 2.53 (m, 2H), 2.13
(q, J=7 Hz, 2H), 1.77 (m, 2H), 1.70 (m, 2H), 1.52 ppm (m, 2H);
13C NMR (150 MHz, [D6]DMSO): d=24.0, 27.5, 28.2, 33.8, 34.0, 59.2,
61.0, 64.2, 67.1, 84.6, 114.3, 129.1, 133.5, 140.9, 156.8 ppm; IR (solid
˜
66.9, 112.7, 114.3, 126.2, 129.1, 133.4, 156.8 ppm; IR (solid film): n=
˜
film): n=2940, 2865, 1620, 1613, 1583, 1541, 1512, 1245, 1176,
3355, 3274, 2937, 2866, 1613, 1582, 1513, 1246, 1178, 1069, 1046,
893, 831 cmꢀ1; LC–MS: tRB =4.64 min; m/z: 420.0 [M+H]; HRMS-
pESI m/z [M+H]+ calcd for C17H27O3NI: 420.10301, found:
420.10287.
1178, 1068, 948, 828 cmꢀ1
; LC–MS. tRB =4.44 min; m/z: 500.1
[M+H]; HRMS-pESI m/z [M+H]+ calcd for C17H28O6NIP: 500.06935,
found: 500.06934.
1
(R/S)-phosphoric acid di-tert-butyl ester 4-{2-[4-((E)-6-iodohex-5-
enyloxy)phenyl]ethyl}-2-methyl-4,5-dihydro-oxazol-4-ylmethyl
ester (10a): 1H-tetrazole (182 mg, 5.0 equiv) and di-tert-butyldi-
ethylphosphoramidite (0.433 mL, 3.0 equiv) were added to a solu-
11 c: H NMR (600 MHz, [D6]DMSO): d=8.26 (brs, 3H), 7.10 (d, J=
7.3 Hz, 2H), 6.83 (d, J=7.9 Hz, 2H), 6.19 (s, 1H), 5.72 (s, 1H), 3.92
(m, 4H), 3.54 (m, 2H), 2.52 (m, 2H), 2.43 (t, J=7.1 Hz, 2H), 1.79 (m,
2H), 1.68 (m, 2H), 1.58 ppm (m, 2H); 13C NMR (150 MHz,
674
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 667 – 677

BZM055
[D6]DMSO): d=25.2, 27.2, 27.4, 33.6, 44.0, 59.2, 60.9, 66.9, 112.7,
hot CH3OH to afford 14 as a white solid (143 mg, 74%). tRA =
1
˜
114.3, 129.1, 133.3, 156.8 ppm; IR (solid film): n=2940, 2866, 1614,
4.35 min; H NMR (600 MHz, [D6]DMSO): d=7.13 (s, 1H), 6.753 (s,
2H), 6.42 (brs, 1H), 4.0–3.96 (m, 2H), 3.57 (d, J=11.5 Hz, 2H), 2.56–
2.52 (m, 2H), 2.40 (t, J=7.17 Hz, 2H), 1.82–1.75 (m, 2H), 1.56–1.48
(m, 2H), 1.43 (s, 9H), 1.34–1.31 (m, 6H), 1.30–1.23 (m, 10H),
0.84 ppm (t, J=6.56 Hz, 3H); 13C NMR (600 MHz, [D6]DMSO): d=
154.52, 142.58, 136.47, 132.22, 127.09, 125.06, 97.37, 77.40, 64.64,
51.72, 35.37, 31.52, 31.32, 28.96, 28.72, 28.27, 25.25, 22.10,
1540, 1512, 1245, 1179, 1141, 1044, 934, 895, 835 cmꢀ1; LC–MS:
t
RB =4.45 min; m/z: 499.9 [M+H]; HRMS-pESI m/z [M+H]+ calcd for
C17H28O6NIP: 500.06935, found: 500.06943.
{5-[2-(2-Iodo-4-octylphenyl)ethyl]-2,2-dimethyl[1,3]dioxan-5-yl}-
carbamic acid tert-butyl ester (12): A mixture of 2a (0.89 g,
2.05 mmol), (Boc)2O (0.67 g, 3.08 mmol) and 1n NaOH (2.26 mL,
2.26 mmol) in dioxane (50 mL) was stirred at RT overnight. The re-
action mixture was extracted with EtOAc, and the combined or-
ganic fractions were dried over Na2SO4, filtered, concentrated and
purified by flash chromatography on silica gel (hexane/EtOAc,
60:40) to give Boc-2a as a colorless oil (905 mg, 83%). 2,2-dime-
thoxypropane (19.9 mL, 159 mmol), acetone (11.7 mL, 159 mmol),
and pTsOH·H2O (30 mg, 0.16 mmol) were added to a solution of
Boc-2a (850 mg, 1.59 mmol) in DMF (15 mL) and stirred at RT for
1 h. The solution was quenched with saturated aqueous NaHCO3,
extracted with EtOAc, and the combined fractions dried over
Na2SO4, filtered and concentrated. The crude product was purified
by flash chromatography on silica gel (hexane/EtOAc, 90:10) to
give 800 mg (88%) 12 as a colorless oil. tRA =4.51 min; 1H NMR
(600 MHz, [D6]DMSO): d=7.62 (s, 1H) 7.19–7.08 (m, 2H) 6.69 (brs,
1H) 3.88–3.84 (m, 2H) 3.71 (d, J=11.71 Hz, 2H) 2.49–2.64 (m, 2H)
2.30–2.47 (m, 2H) 1.94–1.77 (m, 2H) 1.46–1.44 (m, 2H) 1.43–1.37
(m, 9H) 1.36 (s, 3H) 1.34 (s, 3H) 1.16–1.30 (m, 10H) 0.85 ppm (t,
J=6.96 Hz, 3H); 13C NMR (600 MHz, [D6]DMSO): d=154.53, 142.40,
141.72, 138.60, 128.99, 128.73, 100.30, 97.72, 77.73, 64.86, 51.17,
33.88, 33.55, 32.63, 31.26, 30.81, 28.75, 28.62, 28.30, 23.63, 22.08,
˜
13.97 ppm; IR (solid film): n=3332, 2993, 2977, 2959, 2926, 2854,
1700, 1536, 1392, 1368, 1173 cmꢀ1; FTMS-pESI m/z [MꢀH]ꢀ; Anal.
calcd for C27H44O4NBF3: 514.305145, found: 514.33244.
[g-35S]GTP binding assay
Chinese hamster ovary (CHO) cells expressing the human S1P re-
ceptor were harvested in ice-cold buffer [10 mm HEPES (pH 7.5),
10 mm EDTA, and protease inhibitor cocktail (1:50 complete, Roche
Applied Science)], centrifuged at 750 g for 10 min at 48C, and re-
suspended in 20 mm HEPES (pH 7.5), 100 mm NaCl, 1 mm EDTA,
and protease inhibitor cocktail. The cell suspension was homogen-
ized on ice with a Polytron Homogenizer at 25000 rpm, centri-
fuged at 26900 g for 30 min at 48C, and resuspended in 20 mm
HEPES (pH 7.5), 100 mm NaCl, 1 mm EDTA, and protease inhibitor
cocktail at 2–3 mg proteinmLꢀ1 and stored in aliquots at ꢀ808C.
To characterize [g-35S]GTP binding, membrane proteins (10–
25 mgmLꢀ1) were resuspended in assay buffer [20 mm HEPES,
100 mm NaCl, 10 mm MgCl2, 20 mgmLꢀ1 saponin, 10 mm GDP, and
0.1% fat-free BSA (pH 7.4) mixed with 5 mgmLꢀ1 WGA-coated SPA
beads (Amersham Biosciences)] and various concentrations of ago-
nists and incubated for 10–15 min at RT. The [g-35S]GTP binding re-
action was started by the addition of [g-35S]GTP (PerkinElmer,
>1000 Cimmolꢀ1), final concentration: 200 pm. After 120 min incu-
bation at RT, the plates were centrifuged for 10 min at 1200 rpm
(300 g) and counted with a TopCountNXT instrument (Packard
Instruments).
˜
13.98 ppm; IR (solid film): n=3342, 3352, 2956, 2927, 2856, 1718,
1600, 1496, 1454, 1368, 1248, 1200, 1167, 1073, 833 cmꢀ1; UPLC–
MS[SQD] (ES+): m/z 574 [M+H]+.
(2,2-Dimethyl-5-{2-[4-octyl-2-(4,4,5,5-tetramethyl[1,3,2]dioxa-
borolan-2-yl)phenyl]ethyl}[1,3]dioxan-5-yl)carbamic acid tert-
butyl ester (13): A 20 mL vial was charged with 1,1’-bis(diphenyl-
phosphino)ferrocene–PdII dichloride CH2Cl2 complex (8.54 mg,
10,46 mmol), KOAc (103 mg, 1046 mmol), bis(pinacolato)diboron
(97 mg, 0.38 mmol), and flushed with argon. A solution of 12
(200 mg, 0.349 mmol) in DMSO (6 mL) was added, and the solution
was stirred for 4 h at 808C. The reaction mixture was quenched
with H2O and extracted with EtOAc. The combined organic frac-
tions were washed with brine and dried over Na2SO4, then filtered,
concentrated under reduced pressure, and purified by flash chro-
matography on silica gel (hexane/EtOAc, 90:10) to give 13 as a
white powder (137 mg, 68%). tRA =5.02 min; 1H NMR (600 MHz,
[D6]DMSO): d=7.46–7.44 (m, 1H), 7.21 (d, 1H), 7.00 (d, 1H), 6.51
(brs, 1H), 3.92 (d, 2H), 3.69 (d, 2H) 2.72–2.64 (m, 2H), 2.56–2.52
(m, 2H), 1.85–1.78 (m, 2H), 1.58–1.49 (m, 2H), 1.46–1.41 (m, 9H),
1.36–1.34 (m, 6H), 1.32–1.30 (m, 12H), 1.29–1.25 (m, 10H), 0.90–
0.84 ppm (m, 3H); 13C NMR (600 MHz, [D6]DMSO): d=154.54,
146.41, 138.71, 135.58, 131.20, 129.01, 127.25, 97.54, 83.26, 77.70,
64.71, 51.53, 35.85, 34.63, 31.26, 28.65, 28.24, 24.59, 22.08,
Animal studies
Animal work was performed according to the Swiss federal law for
animal protection and approved by the Veterinary Office Basel (BS
No. 1325), with an acclimation period of >1 week before use, con-
ventional hygienic conditions for housing (five animals per cage,
temperature 20–248C, minimum relative humidity 40%, light/dark
cycle 12 h), standard diet (KLIBA Nr. 3893.0.25) and drinking water
ad libitum.
Lymphocyte depletion assay in Lewis rats
The rats (220–250 g males from Charles River, Germany) were treat-
ed i.v. via tail vein with test compound at 4 mgkgꢀ1. All com-
pounds were dissolved in a solution of 0.9% NaCl at 2 mgmLꢀ1 in
order to get an injection volume of 2 mLkgꢀ1. For each treatment
group, rats were anesthetized (5% v/v isoflurane; Foreneꢀ, Abbott,
Baar, Switzerland) at 0.5, 2, 4, 8, 24 and 48 h post-treatment (n=3
per time point) and blood samples (~200 mL) were collected by
sublingual punctures in EDTA-coated Eppendorf tubes to assess
the lymphocyte counts using an automated hematology analyzer
(Technicon H1-E analyzer, Bayer Diagnostics, Zꢄrich, Switzerland).
Serum leftovers were kept frozen at ꢀ808C for latter processing to
assess drug blood levels. The rats were then decapitated, brains
collected, weighted and snap frozen in liquid nitrogen.
˜
13.96 ppm; IR (solid film): n=3331, 3241, 3048, 3031, 2989, 2977,
2923, 2869, 2855, 1706, 1536, 1369, 1339, 1309, 1174, 1148, 1127,
1087, 1068, 858, 847, 831 cmꢀ1; UPLC–MS[SQD] (ES+): m/z 574
[M+H]+.
Preparation of trifluoroboronate precursor: (2,2-dimethyl-5-{2-
[4-octyl-2-(trifluoroborolan-2-yl)phenyl]ethyl}[1,3]dioxan-5-yl)-
carbamic acid tert-butyl ester (14): Aqueous KHF2 (0.44 mL,
1.97 mmol) was added to a solution of 13 (200 mg, 0.35 mmol) in
CH3OH (2 mL). The resulting slurry was stirred at RT for 15 min,
concentrated in vacuo, and dissolved in hot acetone, filtered and
concentrated again in vacuo. The residue was recrystallized from
ChemMedChem 2011, 6, 667 – 677
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
675

E. Briard et al.
MED
mode for the non-phosphorylated species, and in negative mode
for the phosphorylated species with parameters optimized for the
phosphorylated species. Quantification was based on the com-
pound/IS ratio of the extracted ion chromatograms of the selected
mass transitions 560 m/z ! 500 m/z for BZM055 (2a) and 638 m/z
! 536.2+596.3 m/z for 5a. The unknown sample concentration
was calculated using the polynomial fitted (r2 ꢂ0.9962) external
calibration curves. The LLOQ of the method was 10 ngmLꢀ1 for
blood, and 50 ngmLꢀ1 for brain samples, and the recovery from
the matrix was >65%. All calculations were performed with Xcali-
bur 2.0.7 software (Thermo Scientific, Reinach, Switzerland), and PK
calculation and data presentation were done with PK Solution 2.0
software (Summit Research Services, Ashland, OH, USA) and MS
Excel 2007.
Quantitative determination in blood and brain
Sample preparation and analysis was based on a modified protein
precipitation procedure followed by liquid chromatographic sepa-
ration coupled with mass spectrometry for detection. PK parame-
ters were evaluated with MS Excel 2007 and PK Solution 2.0
(Summit Research Sevices, Ashland, OH, USA).
Sample preparation
Prior sample preparation whole brains were homogenized in a Dis-
pomix homogenizer (Milteniy Biotec, Bergisch Gladbach, Germany)
by adding 50% CH3OH to a final concentration of 0.25 gmLꢀ1
Blood samples were directly used for further preparation.
.
Calibration, quality control, and recovery control samples were pre-
pared by spiking blank blood and blank brain homogenate with
known quantities of the parent compound (between 1 and
10000 ngmLꢀ1) and the corresponding phosphate, respectively.
For FTY720 and AML629 determination, the d4-labeled compounds
were used as internal standards (IS), whereas for BZM055 (2a) and
5a, structurally related compounds were used.
Acknowledgements
The authors thank Emanuele Mauro and Samuel Haessig for syn-
thetic support, Beatrice Urban for sample preparation and bio-
analysis, as well as Dr. Eric Francotte, Georg Diehl, Monique Kess-
ler, and Christophe Bury for HPLC and SFC separations.
Aliquots of 100 mL calibration standard, quality control, recovery
control, and unknown samples were transferred to 2 mL Eppendorf
tubes (Vaudaux-Eppendorf, Schçnenbuch, Switzerland) and 25 mL
IS mixture (0.4 mgmLꢀ1 in 50% CH3CN) was added to each tube.
For protein precipitation and extraction from the blood and brain
matrix, 700 mL CH3CN/CH3OH/CHCl3 40:30:30 was added. After
ultrasonication (Bandelin SONOREX RK126 at RT) for 10 min and
vortexing for 15 min, the samples were centrifuged at 16000 g for
5 min at RT. The whole upper layer was transferred to 1.5 mL HPLC
vials (Chromacol Gold, Infochroma AG, Zug, Switzerland) and re-
duced to dryness using a SpeedVac Plus concentrator (Thermo Sci-
entific, Reinach, Switzerland). The residues were acetylated for
20 min at 408C in a water bath by adding 100 mL pyridine and
50 mL Ac2O. All samples were evaporated to dryness in a SpeedVac
concentrator using medium heater temperature. The residues were
re-dissolved by adding 60 mL 0.2% formic acid in CH3OH, then vor-
texing and ultrasonication (Bandelin SONOREX RK126 at RT) for
10 min each. After a short centrifugation step, samples were stored
at 158C in a cooled autosampler until analysis.
Keywords: imaging agents
emission tomography
· iodine · myelin · positron
[1] L. Kappos, E.-W. Radue, P. O’Connor, C. Polman, R. Hohlfeld, P. Calabresi,
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LC–MS–MS analysis
For quantitative analysis, a 10 mL aliquot of each sample, including
calibration, quality control, and recovery control samples were in-
jected with a cooled PAL HTS autosampler (CTC Analytics, Ziefen
Switzerland) on a 2.0ꢃ50 mm column filled with 1.8 mm Gold-
Turbo Basic C18 stationary phase for blood samples and on a
column of the same geometry filled with 2.5 mm Reprosil-Pur C18
stationary phase (Morvay Analytik GmbH, Basel, Switzerland) for
brain samples. Columns were held at 408C (HotDog 5090, Prolab,
Reinach, Switzerland), and for separation a two-step linear gradient
from 5 to 60% B in 1 min and from 60 to 100% B in 5 min at a
flow rate of 0.100 mLminꢀ1 was applied using a Rheos Allegro
pump (Thermo Scientific, Reinach, Switzerland). The total cycle
time was 14 min. The mobile phase used was A: 5 mm ammonium
formate with 0.2% formic acid in H2O, and B: 5% CH3CN in CH3OH.
To minimize carryover, the column and injector were washed
extensively after each injection.
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ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 667 – 677

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Published online on January 30, 2011
ChemMedChem 2011, 6, 667 – 677
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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